Tumor Cells In Specimens Research Articles

DNA methylation-based machine learning classification distinguishes pleural mesothelioma from chronic pleuritis, pleural carcinosis, and pleomorphic lung carcinomas

ObjectivesOur goal was to evaluate the diagnostic value of DNA methylation analysis in combination with machine learning to differentiate pleural mesothelioma (PM) from important histopathological mimics. Material and methodsDNA methylation data of PM, lung adenocarcinomas, lung squamous cell carcinomas and chronic pleuritis was used to train a random forest as well as a support vector machine. These classifiers were validated using an independent validation cohort including pleural carcinosis and pleomorphic variants of lung adeno- and squamous cell carcinomas. Furthermore, we performed differential methylation analysis and used a deconvolution method to estimate the composition of the tumor microenvironment. ResultsT-distributed stochastic neighbor embedding clearly separated PM from lung adenocarcinomas and squamous cell carcinomas, but there was a considerable overlap between chronic pleuritis specimens and PM with low tumor cell content. In a nested cross validation on the training cohort, both machine learning algorithms achieved the same accuracies (94.8%). On the validation cohort, we observed high accuracies for the support vector machine (97.8%) while the random forest performed considerably worse (89.5%), especially in distinguishing PM from chronic pleuritis. Differential methylation analysis revealed promoter hypermethylation in PM specimens, including the tumor suppressor genes BCL11B, EBF1, FOXA1, and WNK2. Deconvolution of the stromal and immune cell composition revealed higher rates of regulatory T-cells and endothelial cells in tumor specimens and a heterogenous inflammation including macrophages, B-cells and natural killer cells in chronic pleuritis. ConclusionDNA methylation in combination with machine learning classifiers is a promising tool to reliably differentiate PM from chronic pleuritis and lung cancer, including pleomorphic carcinomas. Furthermore, our study highlights new candidate genes for PM carcinogenesis and shows that deconvolution of DNA methylation data can provide reasonable insights into the composition of the tumor microenvironment.

Opposite Roles of Tumor Cell Proliferation and Immune Cell Infiltration in Postoperative Liver Metastasis of PDAC.

BackgroundRecurrence of liver metastasis after pancreatectomy is often a predictor of poor prognosis. Comprehensive genomic analysis may contribute to a better understanding of the molecular mechanisms of postoperative liver metastasis and provide new therapeutic targets.MethodsA total of 67 patients from The Cancer Genome Atlas (TCGA) were included in this study. We analyzed differentially expressed genes (DEGs) by R package “DESeq2.” Weighted gene co-expression network analysis (WGCNA) was applied to investigate the key modules and hub genes. Immunohistochemistry was used to analyze tumor cell proliferation index and CD4+ T cells infiltration.ResultsFunctional analysis of DEGs between the liver metastatic and recurrence-free groups was mainly concentrated in the immune response. The liver metastasis group had lower immune and stroma scores and a higher TP53 mutation rate. WGCNA showed that the genes in key modules related to disease-free survival (DFS) and overall survival (OS) were mainly enriched in the cell proliferation process and tumor immune response. Immunohistochemical analysis showed that the pancreatic cancer cells of patients with early postoperative liver metastasis had higher proliferative activity, while the infiltration of CD4+ T cells in tumor specimens was less.ConclusionOur study suggested that increased immune cell infiltration (especially CD4+ T cells) and tumor cell proliferation may play an opposite role in liver metastasis recurrence after pancreatic cancer.

Hyperion Image Analysis Depicts a Preliminary Landscape of Tumor Immune Microenvironment in OSCC with Lymph Node Metastasis.

Background Oral squamous cell carcinoma (OSCC) constitutes the most common types of oral cancer. Because its prognosis varies significantly, identification of a tumor immune microenvironment could be a critical tool for treatment planning and predicting a more accurate prognosis. This study is aimed at utilizing the Hyperion imaging system to depict a preliminary landscape of the tumor immune microenvironment in OSCC with lymph node metastasis. Methods We collected neoplasm samples from OSCC patients. Their formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained and stained utilizing a panel of 26 clinically relevant metal-conjugated antibodies. Detection and analysis were performed for these stained cells with the Hyperion imaging system. Results Four patients met our inclusion criteria. We depicted a preliminary landscape of their tumor immune microenvironment and identified 25 distinct immune cell subsets from these OSCC patients based on phenotypic similarity. All these patients had decreased expression of CD8+ T cells in tumor specimens. Variety in cell subsets was seen, and more immune activated cells were found in patient A and patient B than those in patient C and patient D. Such differences in tumor immune microenvironments can contribute to forecasting of individual prognoses. Conclusion The Hyperion imaging system helped to delineate a preliminary and multidimensional landscape of the tumor immune microenvironment in OSCC with lymph node metastasis and provided insights into the influence of the immune microenvironment in determination of prognoses. These results reveal possible contributory factors behind different prognoses of OSCC patients with lymph node metastasis and provide reference for individual treatment planning.

CBMS-05 Biological and Pathological meaning of aneuploidy in mouse glioma stem sell

Chromosomal instability (CIN) is a pathological condition where cells continuously mis-segregate chromosomes, producing aneuploid cells. CIN has been recognized as a hallmark of cancer, and its correlation with biological malignancy has been pointed out. Glioma cell line often reveals karyotype aberrations, and a variety of ploidy in the tumor tissue. However, several studies have indicated that aneuploidy is disadvantageous for proliferation or tumorigenesis, and these paradox prompt us to address the role of aneuploid cells in tumor specimens. Here, we adopted mouse glioma stem cell lineages and found that aneuploid population is increased in glioma stem cells in vitro. We also examined Aurora B at centromeres which is crucial for failsafe chromosome segregation and found its reduced activity in glioma stem cells, suggesting that insufficient Aurora B activity plays a causative role in CIN in glioma stem cells. Next, to investigate the tumorigenicity of aneuploid cells, we sorted the glioma stem cells depending on the karyotype pattern, and allografted into mouse brain. We found that the growth rate of diploid glioma stem cells was higher than others in vitro, and the probability of survival after allogeneic transplant was significantly lower in diploid groups. We will discuss the role of ploidy in glioma cells populations.

Digital PCR for discriminating mosaic deletions and for determining proportion of tumor cells in specimen.

Mosaicism, presence of a genetic feature in only a subpopulation of cells, is frequent in de novo genetic diseases. Among large deletions covering the NF1 tumor suppressor gene, the frequency of mosaicism can be as high as 40% in de novo patients. In this study, we demonstrate the high potential of digital PCR in detecting large NF1 deletions and in discriminating mosaic cases. By simultaneously assessing the NF1 gene and a reference gene RPP30, deletions could be unambiguously distinguished from non-deletion samples. Performing the same assay for mixed samples from a DNA with a deletion and a non-deletion DNA, a highly significant linear relation was obtained between the set-up ratio of the two samples and the measured ratio of NF1/RPP30 (P<0.0001), suggesting the high potential of digital PCR in discriminating mosaic deletions. Furthermore, digital PCR detects NF1 allele loss in a tumor specimen that was not detected by loss of heterozygosity analysis using polymorphic markers due to high content of non-tumor cells. Based on the measured ratio of NF1/RPP30, the proportion of the tumor cells in this specimen could be calculated as 25%. Our results demonstrate that dual-probe digital PCR is a simple and effective method for detecting deletions and for discriminating mosaic deletions. Furthermore, this method is sensitive for assigning somatic allele loss in tumor specimen and enables determining proportion of tumor cells.

Non-Invasive Detection of Genomic Imbalances in Hodgkin/Reed-Sternberg Cells in Early and Advanced Stage Hodgkin Lymphoma By Sequencing of Circulating Cell-Free DNA

Background. Hodgkin lymphoma (HL) accounts for 11-30 % of all lymphomas and is one of the most common lymphoid neoplasms in adolescents and young adults. HL is highly curable today, but 10-15 % of patients will be resistant to or will relapse after first-line therapy. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells in classical HL (cHL), represent only 0,1-2 % of cells in cHL biopsies, while the remainder is composed of a mixture of non-malignant immune cells. The low abundance of HRS cells severely hampers the identification of recurrent genetic lesions in cHL, as well as the elucidation of its pathogenesis, biology and diversity.Methods. We applied massive parallel sequencing to circulating cell-free DNA (ccfDNA) in a prospective study of patients with biopsy proven nodular sclerosis cHL (NSHL) stage IIA-IVB. Genomic imbalances in HRS cells were investigated by fluorescence in situ hybridization (FISH) on tumor specimens.Results. We recently implemented a novel pipeline for non-invasive prenatal testing in pregnancy. Based on massive parallel sequencing of ccfDNA, it allows genome-wide detection of fetal aneuploidies and segmental imbalances. In one case, a complex profile with several genomic imbalances was identified. After exclusion of fetal and maternal constitutional abnormalities, the possibility of a maternal or fetal tumor was considered, and this led to a biopsy-proven diagnosis of early-stage NSHL stage IIA in the pregnant mother. In order to verify the origin of the genomic imbalances found in ccfDNA, we resorted to FISH analysis of the rare HRS cells in formalin-fixed paraffin-embedded biopsy sections. Matching gains of 8qter, 9pter and 14q were found in HRS cells, which strongly suggested that DNA derived from HRS cells was causing the abnormal ccfDNA profile in this case. To further investigate the recurrence of this phenomenon, ccfDNA was prospectively collected from nine cases with NSHL (age 12-65 y), at first diagnosis (n=8) or at relapse (n=1). Seven patients had stage IIA disease, and two had stage IVB disease. In eight, genomic imbalances were found by massive parallel sequencing of ccfDNA. Reassuringly, the affected regions were already reported previously in the few studies that investigated genomic imbalances in cHL by aCGH on microdissected HRS cells. In addition, we extensively validated the imbalances suggested in ccfDNA profiling by FISH analysis of HRS cells in tumor specimens from all cases. This yielded concordant results in 24 experiments, partially concordant results in two experiments, and discordant results in three experiments only. Combination of FISH with CD30 immunostaining showed the giant cells with abnormal hybridization patterns to be CD30-positive, identifying them unequivocally as HRS cells. Cells with normal hybridization patterns were CD30-negative. The striking overall agreement between the genomic imbalances in ccfDNA and those in HRS cells found by FISH, cogently proves that ccfDNA contains DNA derived from HRS cells. Although profile abnormalities were most pronounced in stage IVB disease, abnormalities were also detected in seven of eight cases with stage IIA disease. All patients, including the pregnant patient, were treated with ABVD-based chemotherapy and all responded as shown by early clinical and imaging evaluation. This was paralleled by rapid normalization of ccfDNA profiles upon therapy initiation in all cases, underscoring the link between the abnormal ccfDNA profiles and the HRS cell burden. Furthermore, this suggests a potential for ccfDNA profiling in the staging and early response monitoring of cHL. The expression of the cell cycle indicator Ki67 and of cleaved caspase-3 was evaluated by immunohistochemistry in tumor biopsies. Ki67 and cleaved caspase-3 were detected in HRS cells in 10 and 6 cases respectively, consistent with unexpectedly high HRS cell turnover.Conclusions. Non-invasive massive parallel sequencing of ccfDNA allows identification of genomic imbalances in HRS cells in early and advanced stage NSHL. The possibility to interrogate the genomic status of HRS cells in ccfDNA opens important new perspectives for the exploration of the biology of cHL as well as for the diagnosis and management of early and advanced cHL. This novel discovery will facilitate the development of biomarkers and the design of clinical trials with novel biological agents, and may advance targeted and precision therapy in cHL. DisclosuresNo relevant conflicts of interest to declare.

Different mutational characteristics of TSG in cell lines and surgical specimens.

One of the most crucial concerns of cancer research pertains to the differences between the neoplastic cells in tumor specimens in vivo and their counterparts in cell lines. The huge amount of results deposited in cancer genetic databases allows to address this issue from a wider perspective. Our analysis of the Sanger Institute Catalog Of Somatic Mutations In Cancer (COSMIC) database v61 showed a lower percentage of homozygous mutations in a group of tumor suppressor genes in surgical samples (in vivo) in comparison to their frequency in cell lines (in vitro). Similarly, the mutations resulting in the lack of protein (e.g., nonsense mutations or whole gene deletions) of several tumor suppressor genes (TSGs) were more frequently observed in vitro than in vivo. In this article, we suggest two potential explanations of these data. Firstly, TSG heterozygous mutations resulting in the modified protein (e.g., missense mutations) may be gradually (when the specific molecular context is achieved) changed to homozygous mutations resulting in the lack of protein during carcinogenesis. Secondly, among different independent pathways of tumorigenesis, those leading to homozygous nonsense mutations are characteristic for cells which are more efficiently stabilized in vitro. To conclude, these observations may be interesting for researchers working with cell line in vitro models illustrating the extent to which they reflect the tumors in vivo.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-014-2444-5) contains supplementary material, which is available to authorized users.

The Ratios of CD8+ T Cells to CD4+CD25+ FOXP3+ and FOXP3- T Cells Correlate with Poor Clinical Outcome in Human Serous Ovarian Cancer

Ovarian cancer is an immune reactive malignancy with a complex immune suppressive network that blunts successful immune eradication. This suppressive microenvironment may be mediated by recruitment or induction of CD4+ regulatory T cells (Tregs). Our study sought to investigate the association of tumor-infiltrating CD4+CD25+FOXP3+ Tregs, and other immune factors, with clinical outcome in serous ovarian cancer patients. We performed immunofluorescence and quantification of intraepithelial tumor-infiltrating triple positive Tregs (CD4+CD25+FOXP3+), as well as CD4+CD25+FOXP3-, CD3+ and CD8+ T cells in tumor specimens from 52 patients with high stage serous ovarian carcinoma. Thirty-one of the patients had good survival (i.e. > 60 months) and 21 had poor survival of < 18 months. Total cell counts as well as cell ratios were compared among these two outcome groups. The total numbers of CD4+CD25+FOXP3+ Tregs, CD4+CD25+FOXP3-, CD3+ and CD8+ cells were not significantly different between the groups. However, higher ratios of CD8+/CD4+CD25+FOXP3+ Treg, CD8+/CD4+ and CD8/CD4+CD25+FOXP3- cells were seen in the good outcome group when compared to the patients with poor outcome. These data show for the first time that the ratios of CD8+ to both CD4+CD25+FOXP3+ Tregs and CD4+CD25+FOXP3- T cells are associated with disease outcome in ovarian cancer. The association being apparent in ratios rather than absolute count of T cells suggests that the effector/suppressor ratio may be a more important indicator of outcome than individual cell count. Thus, immunotherapy strategies that modify the ratio of CD4+CD25+FOXP3+ Tregs or CD4+CD25+FOXP3- T cells to CD8+ effector cells may be useful in improving outcomes in ovarian cancer.

Expression of Leptin and iNOS in Oral Melanomas in Dogs

Oral melanoma (OM) in dogs is an aggressive malignancy, with clinical behavior resembling cutaneous melanomas in humans. Melanoma in humans is promoted by an inflammatory environment that is contributed to by leptin and inducible nitric oxide synthase (iNOS). To determine if the patterns of leptin and iNOS expression are similar in OM in dogs and cutaneous melanomas in humans. Twenty client-owned dogs. Retrospective case study. Immunostaining of the OM tumors from each dog was scored for percentage and intensity of leptin and iNOS expression. Mitotic index was used as an indicator of tumor aggression. Leptin was detected in ≥75% of the tumor cells in specimens from 11 dogs. One tumor expressed leptin in ≤25% of the cells. The intensity of leptin expression was variable with 6, 9, and 5 cases exhibiting low-, moderate-, and high-intensity staining, respectively. OM with the lowest percentage of iNOS positive cells displayed the highest mitotic indices (P = .006, ANOVA). The expression of leptin is a common finding in melanomas in dogs. These data suggest that the possibility of future clinical applications, such as measuring the concentrations of plasma leptin as a screening tool or leptin as a target for therapy. The relevance of iNOS is not as clear in dogs with OM, for which other directed therapeutics might be more appropriate.

Abstract 1258: The frequency of CD11c+ cells within the gastric cancer tissue is associated with favorable prognosis.

Abstract Aim: CD11c is a marker for dendritic cells which help initiating adaptive antitumor immunity. The study was designed to investigate correlation between the frequency of CD11c+ cells in the gastric cancer tissue and the clinical and pathological parameters of gastric cancer patients. Methods: CD11c expression in gastric cancer tissues was examined by immunohistochemistry. Surface CD11c expression on the leukocytes within cancer tissues was examined by flow cytometry. Results: The number of CD11c+ cells was higher in gastric cancer tissues than that in gastritis or gastric polyps tissues. The number of CD11c+ cells in tumor specimens from the early-stage gastric cancer was significantly higher than that in specimens from the late-stage gastric cancer (t=2.77,P=0.0064). Flow cytometry found that CD11c surface expression was stronger in specimens from the early stages than those from the late stages of gastric cancer. The Univariate analysis revealed that the frequency of intratumoral CD11c+ cells showed association with gender and tumor location, and was reversely correlated with tumor size, depth of infiltration, nodal metastasis, tumor histological grade and cancer stages. The median survival time (MST) for the subgroups of patients with stronger and weaker CD11c expression were 67.0 months (95%CI, 39.0-112.0 months) and 29.0 months (95%CI, 21.0-39.0 months), respectively. And MSTs were significantly different between the CD11c higher and lower subgroups (χ2=11.6425,P=0.0006). Among patients who suffered from late-stage diseases, those with higher numbers of intratumoral CD11c cells had a significantly lower risk of death than those with lower numbers of CD11c cells (χ2=8.549,P=0.0035), and the lowered risk of death was statistically significant after adjusting gender, age, tumor size and infiltrating depth (RR=0.52,95%CI=0.29-0.94). Conclusions: The number of CD11c+ cells within gastric cancer tissue is a favorable prognostic marker for gastric cancer. Our study suggests gradual elimination of dendritic cells is characteristic of gastric cancer progression. Citation Format: Jingting Jiang, Changping Wu, Binfeng Lu. The frequency of CD11c+ cells within the gastric cancer tissue is associated with favorable prognosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1258. doi:10.1158/1538-7445.AM2013-1258 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell differentiation

The G protein-coupled formylpeptide receptor (FPR), known to mediate phagocytic leucocyte chemotaxis in response to bacterial- and host-derived agonists, was expressed by tumor cells in specimens of surgically removed more highly malignant human gliomas. In human glioblastoma cell lines, FPR activation increased cell motility, tumorigenicity and production of angiogenic factors. In studies of the mechanistic basis for the selective expression of FPR in more highly malignant gliomas, we found that the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), while promoting the differentiation of human glioblastoma cells, downregulated FPR expression. Aza also reduced the global methylation levels in glioblastoma cells and activated the pathway of p53 tumor suppressor. Methylation-specific polymerase chain reaction revealed that Aza treatment of tumor cells reduced the methylation of p53 promoter, which was accompanied by increased expression of p53 gene and protein. In addition, overexpression of p53 in glioblastoma cells mimicked the effect of Aza treatment as shown by increased cell differentiation but reduction in FPR expression, the capacity of tumor sphere formation in soft agar and tumorigenesis in nude mice. Furthermore, Aza treatment or overexpression of the wild-type p53 in glioblastoma cells increased the binding of p53 to FPR promoter region shown by chromatin immunoprecipitation. These results indicate that increased methylation of p53 gene retains human glioblastoma cells at a more poorly differentiated phase associated with the aberrant expression of FPR as a tumor-promoting cell surface receptor.

Monitoring patients for bladder neoplasms: what can be expected of urinary cytology consultations in clinical practice

Objectives. To determine what practitioners could reasonably expect from urinary cytology (UC) consultations by analyzing their role in our clinical practice. Methods. Reports of 227 consecutive interpretations on 130 patients collected during a 13-month period were correlated with all available follow-up information. Results. In our practice, UC consultations can predict the presence of bladder carcinoma in nearly 90% of patients, the major mitigating factor being the absence of tumor cells in specimens from patients harboring bladder neoplasms. High-grade neoplasms are most reliably detected with UC. Very low grade neoplasms are difficult to detect, primarily because the cells of these lesions lack features of carcinoma. Significant interobserver variation did not occur. Conclusions. Pathology consultations based on UC can be associated with high diagnostic yields provided that certain factors are understood. These include that (a) the clinical import of diagnostic terms may vary among observers and should be mutually understood in individual practice settings; (b) UC specimens may not contain tumor cells even when patients have bladder cancer; (c) correlative information may be difficult to obtain and thus seem to inflate calculations for false-positive results; (d) interobserver variation can be reduced if cytopathologists use limited and uniform diagnostic terms; and (e) a high level of diagnostic expertise from cytopathologists should be expected.

Sensitive and specific cytokeratin 18 reverse transcription-polymerase chain reaction that excludes amplification of processed pseudogenes from contaminating genomic DNA

Processed pseudogenes of residual contaminating genomic DNA interfere with a sensitive detection of cytokeratin 18 (CK18) mRNA by reverse transcription and polymerase chain reaction (RT-PCR). This may cause false-positive results when CK18 mRNA is used as a marker for ectopic tumor cells in specimens from cancer patients. To establish a sensitive CK18 RT-PCR by excluding the amplification of processed pseudogenes, the following strategy was chosen: (a) CK18 pseudogene sequences were cloned from genomic DNA by PCR; (b) cDNA-specific primers were designed on the basis of mismatches between pseudogenes and cDNA; (c) PCR conditions were adjusted to reach maximum sensitivity and specificity. Epithelial cells (1-10) could be detected in 1 mL of blood. Among the numerous CK18 genes homologous to the transcribed gene, at least two different processed pseudogenes exist that are highly homologous to each other and to the exons of the transcribed CK18 gene.

Cytokeratins as markers of initial stages of squamous metaplasia in feline mammary carcinomas

Summary Expression of keratins (cytokeratins, ck) known to be suitable markers for different types of epithelial differentiation was analyzed in specimens of feline mammary tissue. A panel of specific anti-ck monoclonal antibodies (MAb) was used to determine ck distribution pattern in normal feline tissues (n = 3), and in benign (n = 18) and malignant (n = 20) feline mammary tumors. In selected tumors, the ck distribution pattern was also determined by biochemical methods. A MAb specific for α-smooth muscle actin was used to discriminate between myoepithelial cells and luminal epithelial cells. In normal mammary gland tissues, 6 MAb reacted exclusively, either with myoepithelial cells or with luminal epithelial cells. Luminal epithelial cells reacted with MAb specific for ck typical of simple epithelia, whereas myoepithelial cells reacted with MAb specific for ck in basal cells of stratified epithelia. A similar distribution of ck was detected in specimens from benign tumors, except that ck4 was not detected in normal mammary gland tissues and was detected in some ducts in specimens with adenosis. Almost all tumor cells in specimens from malignant tumors reacted with MAb specific for ck typical of simple epithelia. Concomitant expression of ck typical of stratified epithelia was detected in small or large subpopulations of tumor cells in 70% of carcinomas. Cytokeratins typical of basal cell layers and typical for suprabasal layers of inner stratified epithelia were detected. Cytokeratins typical of stratified epithelia were always found in areas of squamous metaplasia, but also were found in adenocarcinomal cells surrounding these areas. We postulate that these adenocarcinoma cells represent transitional cells where the initial stages of squamous metaplasia have taken place, but squamous metaplasia is not yet detectable by light microscopy.

Flow cytometry DNA analysis on tumor cell subpopulation of human tumor specimens by exclusion of lymphohemopoietic cells.

We describe a new flow cytometry procedure in which DNA analyses can be obtained selectively on pure, freshly obtained tumor cell subpopulations of human tumor specimens. This procedure is based on exclusion from analysis of the contaminating lymphohemopoietic cells mixed with tumor cells in tumor specimens. This exclusion is made possible by labeling all lymphohemopoietic cells with an antibody to HLe-1 (HLE), which is present on all lymphohemopoietic cells but on no other cells, and by gating against these labeled cells when analyzing for DNA. For the model system, a 1:1 mixture of normal human peripheral blood leukocytes and either of two human cancer cell lines, HEp-2 and MCF-7, normal leukocyte contamination can be reduced to 3.1% while retaining 94.7% of tumor cells for DNA analysis. Four examples of human tumor samples, two cases each of malignant effusions and lymph node metastases, were analyzed with this procedure. The results clearly indicate that this new method will improve ploidy analysis/aneuploidy detection and will make it possible to obtain more accurate cell-cycle analyses of tumor cells than have previously been possible. This new procedure will contribute to clinical and biological studies involving DNA of human tumors.